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At least 379 records · Page 21Linked to original sources
PubMed1999

In vivo detection, RNA-binding properties and characterization of the RNA-binding domain of the p7 putative movement protein from carnation mottle carmovirus (CarMV).

Biochemical and structural characterization studies on the p7 putative movement protein from a Spanish isolate of carnation mottle carmovirus (CarMV) have been conducted. The CarMV p7 gene was fused to a sequence coding for a six-histidine tag and expressed in bacteria, allowing the purification of CarMV p7 and the production of a specific antiserum. This antiserum led to the immunological identification of CarMV p7 in infected leaf tissue from the experimental host Chenopodium quinoa. Putative nucleic acid-binding properties of the CarMV p7 have been explored and demonstrated with both electrophoretic mobility shift and RNA-protein blot in vitro assays using digoxigenin-labeled riboprobes. CarMV p7 did not show preferential binding to any of the different regions of the CarMV genomic RNA tested, suggesting that RNA binding was sequence nonspecific. Quantitative analyses of the data allowed calculation of the apparent dissociation constant of the p7-RNA complex (Kd approximately 0.7 microM) and supported a cooperative type of binding. A small 19-amino-acid synthetic peptide whose sequence corresponds to the putative RNA-binding domain of CarMV p7, at the basic central part of the protein, was synthesized, and it was demonstrated that it binds viral RNA probes. Peptide RNA binding was as stable as p7 binding, although data indicated it was not cooperative, thus suggesting that this cooperative binding requires another motif or motifs within the p7 amino acid sequence. The peptide could be induced to fold into an alpha-helix structure in which amino acids that are conserved among carmovirus p7-like proteins are distributed on one side. This alpha-helix motif could define a new and previously uncharacterized RNA-binding domain for plant virus movement proteins.

Amino Acid Sequence↗
PubMed1976

Effect of Fv-1 gene product on synthesis of N-tropic and B-tropic murine leukemia viral RNA.

The production of virus and the synthesis of virus-specific RNA has been studied in Fv-1n/n (NIH/3T3, SIM) and Fv-1b/b (BALB/3T3, SIM-R) cell lines after infection with N- or B-tropic MuLV. It was found that virus production, measured by reverse transcriptase activity in the medium, was 70-100 fold lower in cells resistant at the Fv-1 locus than in permissive cells. The virus-specific RNA, detected by hybridization. In RNA excess with complementary DNA, was reduced by approximately 70-100 fold in cytoplasm of resistant cells compared to permissive cells. A reduction of the same magnitude was observed in the levels of virus-specific RNA extracted from nuclei of resistant cells. Our data therefore show that virus-specific RNA levels are reduced in cells nonpermissive at the Fv-1 locus, suggesting that restriction of the Fv-1 gene product occurs at the level of transcription of the viral genome or at a pre-integration step, or, alternatively, that the RNA transcripts are rapidly degraded after their synthesis.

Cell Line↗
PubMed1994

Differential expression in blood stages of the gene coding for the 21-kilodalton surface protein of ookinetes of Plasmodium berghei as detected by RNA in situ hybridisation.

The developmentally regulated transcription of the gene encoding the ookinete surface protein, Pbs21, has been investigated in the rodent malaria parasite, Plasmodium berghei, by RNA in situ hybridisation using fluorescently labelled DNA probes. We used a procedure that will allow the visualisation of cytoplasmic mRNA in the parasite and of high copy DNA repeats in the nucleus. Specific hybridisation to Pbs21 mRNA occurred in the cytoplasm of female gametocytes, zygotes and ookinetes, while asexual blood stages, male gametocytes and gametes showed no fluorescence. Analysis of the transcription of the Pbs21 gene during blood stage development in two tightly synchronised parasite clones using the same methodology revealed that transcription is restricted to sexual stages and is initiated in immature gametocytes at 19 h post invasion (hpi). At this point in development it is not yet possible to discriminate between the morphology of asexual trophozoites and immature gametocytes. At 24 hpi approximately 50% of the gametocytes transcribed the Pbs21 gene and the morphology of these gametocytes was identical and female. The distribution of the mRNA encoding Pbs21 confirmed that post-transcriptional control of expression occurred in the cytoplasm by repression of translation and not through delayed transport of the message to the cytoplasm. The transcription of the Pbs21 gene is the earliest demonstrated event in gametocytogenesis in rodent malaria species to date.

Animals↗
PubMed1996

Failure to detect paramyxovirus RNA in the skin of connective tissue disease.

Etiology of connective tissue disease is unknown. The association of infectious agents has been suspected serologically, ultrastructurally and recently by means of molecular biological techniques. We extracted RNA from lesions of 25 discoid lupus erythematosus, 9 systemic LE, and 3 systemic sclerosis biopsies, and reverse transcription-polymerase chain reaction was performed for all paramyxoviruses known to infect human beings. None of the samples yielded positive signal for any of the viruses. The existence of paramyxovirus in the skin of connective tissue disease is very unlikely.

Base Sequence↗
PubMed2024

Mudskipper detects combinatorial RNA binding protein interactions in multiplexed CLIP data.

The uncovering of protein-RNA interactions enables a deeper understanding of RNA processing. Recent multiplexed crosslinking and immunoprecipitation (CLIP) technologies such as antibody-barcoded eCLIP (ABC) dramatically increase the throughput of mapping RNA binding protein (RBP) binding sites. However, multiplex CLIP datasets are multivariate, and each RBP suffers non-uniform signal-to-noise ratio. To address this, we developed Mudskipper, a versatile computational suite comprising two components: a Dirichlet multinomial mixture model to account for the multivariate nature of ABC datasets and a softmasking approach that identifies and removes non-specific protein-RNA interactions in RBPs with low signal-to-noise ratio. Mudskipper demonstrates superior precision and recall over existing tools on multiplex datasets and supports analysis of repetitive elements and small non-coding RNAs. Our findings unravel splicing outcomes and variant-associated disruptions, enabling higher-throughput investigations into diseases and regulation mediated by RBPs.

RNA-Binding Proteins↗
PubMed1983

Ultrastructural detection of RNA: complementarity of high-resolution autoradiography and of RNAase-gold method.

The recently developed RNAase-gold cytochemical method and the more classical high-resolution autoradiography following incorporation of tritiated uridine, were applied for the localization of RNA molecules in thin sections of isolated liver cells cultured under control conditions or submitted to drugs known to alter the distribution of nuclear RNA. The similar pattern of labeling obtained with both techniques under the three experimental conditions studied (control, treatments with CdCl2, or actinomycin D), together with the results obtained after RNAase digestion, are a good indication of the high specificity and sensitivity of the RNAase-gold method and provide a demonstration of the complementarity of these two methods for the study of the ultrastructural distribution of nuclear RNA.

Animals↗
PubMed1983

Detection of RNA complementary to herpes simplex virus in human oral squamous cell carcinoma.

Biopsy specimens from patients with oral squamous cell carcinoma were examined by in situ hybridisation for evidence of RNA complementary to herpes simplex virus (HSV) type 1, HSV type 2, and adenovirus type 2. RNA complementary to HSV was found in 66% of carcinomas and 33% of non-malignant lesions from other patients. In a further study with internally paired controls, RNA complementary to HSV was found in 53% of carcinoma biopsy specimens but in no biopsy specimens of normal oral mucosa from the same patients. RNA complementary to HSV was found in over 50% of oral squamous cell carcinomas.

Adenoviruses, Human↗
PubMed1999

A comparison of gel-based, nylon filter and microarray techniques to detect differential RNA expression in plants.

An initial application of plant genomics has been to monitor gene expression on a scale much larger than previously possible. Although multiplexed assays of RNA abundance have developed more quickly than those for protein and metabolite levels, some combination of these approaches will soon be providing our best views yet into plant molecular biology. Three techniques that have made contributions to the RNA transcript portion of this combination are reviewed. Currently, each can produce a profile of expression levels for a large but incomplete set of plant genes, at reproducibly high levels of accuracy and over a range of labor and financial expenses.

Blotting, Northern↗
PubMed2004

MSARI: multiple sequence alignments for statistical detection of RNA secondary structure.

We present a highly accurate method for identifying genes with conserved RNA secondary structure by searching multiple sequence alignments of a large set of candidate orthologs for correlated arrangements of reverse-complementary regions. This approach is growing increasingly feasible as the genomes of ever more organisms are sequenced. A program called msari implements this method and is significantly more accurate than existing methods in the context of automatically generated alignments, making it particularly applicable to high-throughput scans. In our tests, it discerned clustalw-generated multiple sequence alignments of signal recognition particle or RNaseP orthologs from controls with 89.1% sensitivity at 97.5% specificity and with 74.4% sensitivity with no false positives in 494 controls. We used msari to conduct a comprehensive scan for secondary structure in mRNAs of coding genes, and we found many genes with known mRNA secondary structure and compelling evidence for secondary structure in other genes. msari uses a method for coping with sequence redundancy that is likely to have applications in a large set of other comparison-based search methods. The program is available for download from http://theory.csail.mit.edu/MSARi.

Algorithms↗
PubMed1998

Cloning and expression of the Campylobacter jejuni lon gene detected by RNA arbitrarily primed PCR.

Fingerprinting of RNA by arbitrarily primed PCR was used to identify a heat-inducible gene in Campylobacter jejuni. Comparing RNA fingerprints from C. jejuni cells before and after 20 min of heat shock at 48 degrees C, a differentially amplified PCR product was identified which displayed a high degree of homology to bacterial lon genes. By screening C. jejuni genomic libraries, the entire lon gene was cloned and sequenced. It encodes a protein of 791 amino acids with a calculated molecular mass of 90.2 kDa. Alignment of the Lon amino acid sequence with that of other bacterial species revealed an overall identity of up to 56.6% (Helicobacter pylori). Northern and RNA dot blot experiments confirmed heat induction of the C. jejuni lon gene, revealing a maximum 6-8-fold increase in the level of specific mRNA.

ATP-Dependent Proteases↗